Project Summary. The late genes of the papillomaviruses are transcribed throughout all stages of its life cycle. Paradoxically, late gene expression at the mRNA and protein level is restricted to late stages of keratinocyte differentiation when infectious virions are being produced. Although the molecular basis of this restriction is not known, a plausible contributory factor is the Negative Regulatory Element (NRE) found in the viral late gene's 3'UTR. The NREs of Bovine Papillomavirus 1 (BPV1) and human papillomavirus 16 (HVP16) inhibit late gene expression in reporter-based assays in both primary keratinocytes (the natural host cell) or in various cell lines. The BPV1 NRE inhibits by binding to the Ul snRNP splicing factor resulting in inhibition of polyA tail addition. Without a polyA tail, the viral late gene mRNA never matures and is presumably degraded by the nuclear exosome. In contrast to BPV1, the HPV16 NRE is far less understood and is more complicated because it consists of 4 weak, overlapping Ul snRNP binding sites linked to a downstream GU rich region of unknown function. This proposal has two overall goals, first to characterize the HPV16 NRE mechanistically and second to determine its importance to the viral life cycle within the natural environment of differentiating keratinocytes. It is important to note that the regulation of HPV16 genes at the RNA processing level is strikingly understudied when compared to the transcriptional level. The successful completion of this work will rigorously test the importance of the NRE to the viral life cycle and will lead to a mechanistic understanding of NRE function. Relevance. It is estimated that about 400,000 new cases of cervical carcinoma occur worldwide every year, and it is the second leading cause of cancer-related deaths among women after breast cancer. Human papillomavirus (HPV) DNA is found in over 90% of cervical cancers and is also prevalent in anogenital cancers. Epidemiologic and laboratory studies have shown a causative role for HPV and HPV 16 in these cancers. For this reason the molecular biology of the HPV 16 strain has been intensely studied. If this proposal successfully identifies new proteins that regulate viral late gene expression then new therapeutic approaches that disrupt the viral life cycle by disrupting these regulatory proteins can be pursued. Diagnostic tests, based on differential expression of these regulatory proteins, can also be developed. [unreadable] [unreadable] [unreadable]